Technologies for Downstream Processing

Biologics comprise a huge variety of different molecules, activities, and applications and mostly try to mimic functions of natural molecules. Generally, they are of a rather complex nature and often consist of different functions on the same molecule (e.g., Fc receptor and antigen-recognition site of an antibody). Typically, proteins can present different possible modifications, such as glycosylation, disulfide bridges, specific carbon and nitrogen terminal groups, or they have been chemically modified, such as pegylated or conjugated proteins. Biologics may be based on carbohydrates (e.g., the meningitis vaccine), peptides (e.g., insulin), lipid structures, parts of microorganism cell walls, complete cells (e.g., stem-cell treatments, or Bacillus Calmette-Guérin (tuberculosis vaccines), or nucleic acids (e.g., plasmids and genome DNA for gene therapy). Furthermore, the active substance or complex does not consist of one molecule, but a mixture of different ones, such as congeners and isoforms. The ratio of each molecule within the mixture of the family can be crucial for activity. The importance of this ratio becomes more and more visible as analytical methods advance.

ADVANCES IN UPSTREAM PROCESSING

This extremely large variety of molecules necessitates a broad range of production systems, and analytical characterization and quantification methods. Most first-generation biological products were extracted from natural sources, such as animal tissue, human plasma, and wild stem microorganisms, and further separated with basic techniques (e.g., precipitation, centrifugation, and filtration). However, because of the risk of contamination and incompatibility, these traditional techniques have been replaced by more advanced biotechnologies, such as recombinant techniques or methods that can significantly reduce the potential level of contaminants. These methods include solvent, heat, gamma irradiation and microwave treatments, nanofiltration, and anion exchange chromatography. Initial hybridoma monoclonal antibodies or fusion proteins have also been replaced by humanized ones, and the third generation of biologics now focuses on further defined structures with less microheterogeneities and fewer aggregates to avoid adverse immune reactions. Another focus of new technologies is the expression of antibodies or antibody fragments that provide certain functions by, for example, being linked to cell-killing antibody drug conjugates.

In the 1990s and the early 2000s, much progress was made in biological upstream processing with productive high-expression systems, effective clone selection, defined culture media, process intensification and single-use components. Progress also was made with regard to improvements in analytical methods, which were developed to further characterize and quantify molecules in terms of structure, identity, activity, purity, microheterogeneity, quantification, and safety. These advances were crucial for downstream development because they helped to further characterize starting materials, thereby providing more insight into quality and mass balance during processing. As such, target specifications are now stricter and more demanding compared with early biologics.

OVERCOMING DOWNSTREAM CHALLENGES

These upstream developments create challenges in downstream processing to ensure that the biologics achieve the new quality and safety specifications. Other challenges for downstream processing include treating high upstream quantities in time, integrating new chemistries, purifying molecules in a contained system that enables the handling of antibody drug conjugates, and, of course, ensuring that processes are cost effective. Recent developments in the downstream area, however, are helping to overcome these challenges.

Process analytical technologies (PAT)

The development of more effective analytical methods has enabled better control over critical quality and quantity attributes online and atline during downstream processing. FDA encourages the use of these technologies and defines them as "a system for designing, analyzing, and controlling manufacturing through timely measurements (i.e. during processing) of critical quality and performances attributes of raw and in-process materials and processes with the goal of ensuring final product quality" (1).

Cycle time, sample preparation, and the nature of the analytical method are key to determining whether on-line or at-line measurement can be developed. Several other factors also need to be considered when implementing a PAT solution. These factors include the following: the design of the fluid pathway; communication between the measuring software and process software; knowledge about the analytical methods' stability, reproducibility, repeatability and precision; linearity; and limits of quantification combined with processcontrol needs. It also is important to determine whether the techniques should be applied only in process development or in the production of commercial material as well. Systems for in-line dilution (ILD) of buffers or clean-in-place (CIP) solutions are examples that have been installed at the industrial scale. The principle of an ILD system is the preparation of buffers and cleaning solutions by using concentrated stock solutions that are diluted with water to the desired final concentration. These systems must be robust to compensate for the pressure changes of the water preparation loops, backpressure from process units (e.g., tangential flow filtration (TFF) or chromatography), and changes in buffer composition and dilution rates. The principle of ILD is shown in Figure 1.

Depending on user requirements, different technical solutions can be used for the dilution itself (e.g., pumps coupled with mass flow meters, analogue or proportioning valves) to ensure that the inlet pressure is adequately controlled, and the control of the composition after the dilution (e.g., conductivity feedback control on the pumps or valves). Cost analysis has shown that these rather simple solutions can save up to 20% in capital expenditure for new downstream plants (2).

Another example of PAT application concerns atline monitoring and product quality control during the chromatographic elution of a mixture of lipidic compounds, which ensures that the correct chromatographic fractions are selected. In one particular case, because the compounds are not ultraviolet active, a light-scattering method was developed to follow the elution profile. In a second step, an analytical method based on mass spectrometry was implemented to control the composition of the different lipids. The work commenced with off-line analyses at bench scale and continued with experimental at-line analyses, including split streams. After the initial laboratory-scale phase, the process and analytical tools were scaled up to find the optimal technical solution for implementation in an industrial cGMP environment. Figure 2 presents the evaporating lightscattering detection (ELSD) at-line measurement, coupled with a preparative high-performance chromatography (HPLC) system, which was used in the study. A very small part of the elution stream that comes from the HPLC is directed to a splitter and transferred to the light-scattering detector. Because organic solvents are used, a nitrogen purge box is necessary. Using this solution, the time for in-process sample analysis was reduced to one day, compared with approximately two weeks with initial analytical methods. The investment and the time of implementation and validatioin need to be balanced with the gain of operational time.